Low temperature preservation of biological tissues such as organs, i.e., cryopreservation, has been the subject of much research effort. Cryopreservation may be approached by freezing or by vitrification. If the tissue is frozen, ice crystals may form within the tissue that may mechanically disrupt its structure and thus damage its ability to function correctly when it is transplanted into a recipient. Organized tissues are particularly susceptible to mechanical damage from ice crystals formed during freezing.
Cryopreserved human and animal tissues are used in a variety of medical applications. In particular, transplantation of cryopreserved heart valves (allografts) represents a well-established valve replacement option. See O'Brien, M. F., Harrocks, S., Stafford, E. G. et al., J Heart Valve Dis 10:334-344 (2001).
However, application of this treatment concept is limited by degeneration and long-term failure, and complicated due to expensive preservation, storage and shipping infrastructures. See Mayer, J. E. Jr., Sem Thorac Cardiovasc Surg 7:130-132 (1995).
Even when all cryopreservation variables are controlled, there is a limit, which is largely a function of tissue volume and geometry (including any associated fluids and packaging), beyond which traditional cryopreservation methods do not consistently work. For example, in cryopreserved allograft heart valves, the leaflet fibroblasts survive well (70-90%), but neither the endothelial cells nor the smooth muscle cells of the aortic tissue associated with the valve survive. The problems include ice formation either within the cells, the extracellular matrix, the capsule, or, as in the case of heart valve endothelium, compression in the lumen of the associated artery.
Transplantation of allograft heart valves was first clinically introduced in 1962. See Ross, D., Lancet 12:487 (1962). Allograft heart valves have been shown to demonstrate exceptionally good initial hemodynamic characteristics, hardly any thromboembolic events without anticoagulation and better resistance to endocarditis compared to bioprosthetic or mechanical valve substitutes. See O'Brien, M., Stafford, E., Gardner, M., Pohlner, P., McGiffin, D., J Thorac Cardiovasc Surg 94:812-23 (1987); see also Tuna, I. C., Orszulak, T A, Schaff, R V., Danielson, G. K., Ann Thorac Surg 49:619-24 (1990).
Initially, the valves were collected and immediately transplanted as so-called homovitals. See Gonzalez-Lavin, L., McGrath, L B., Amini, S., Graf, D., J Card Surg 3:309-12 (1988). Due to logistic issues, grafts were subsequently stored at 4° C. in tissue culture medium with antibiotics for up to 6 weeks prior to implantation. See Jonas, R. A., Ziemer, G., Britton, L., Armiger, L. C., J Thorac Cardiovasc Surg 96:746-55 (1988).
Eventually, in order to enable long-term storage and improve safety by means of microbiology and virology, cryopreservation with controlled rate freezing and storage in vapor phase nitrogen was introduced. See Watts, L. K., Duffy, P., Field, R. B., Stafford, E. G., O'Brien, M. F., Ann Thorac Surg, 21:230-6 (1976). For the last 20 years frozen cryopreservation (FC) has been the worldwide choice for preservation of human heart valves. See Standards for Tissue Banking, 11th Edition, American Association of Tissue Banks, 2006.
The FC methods employed a strategy where ice formation was encouraged because ice formation is a critical part of the freezing process. See Schenke-Layland, K., Madershahian, N., Riemann, I. et al., Ann Thorac Surg 81:918-26 (2006). Long-term function of cryopreserved heart valves (allografts) is limited by immune responses, inflammation, subsequent structural deterioration and an expensive infrastructure. See Mitchell, R. N., Jonas, R. A., Schoen, F. J., J Thorac Cardiovasc Surg 115:118-27 (1998); see also; Mayer, J. E. Jr., Sem Thorac Cardiovasc Surg 7:130-132 (1995)
The durability of contemporary cryopreserved allograft valves varies from 50% to 90% at 10 to 15 years. See O'Brien, M. F., Harrocks, S., Stafford, E. G. et al., J Heart Valve Dis 10:334-344 (2001). In particular, in pediatric patients allograft function is limited by earlier and faster structural deterioration necessitating more frequent re-intervention procedures. Bonhoeffer, P., Boudjemline, Y., Saliba, Z. et al., Lancet 356:1403-5 (2000); see also Joudinaud, T. M. et al., Eur J Cardiothorac Surg 33:989-94 (2008). A variety of mechanisms for this increased structural deterioration have been proposed including T-cell mediated inflammation. See Legare J F, Lee T D, Creaser K, Ross D B, Ann Thorac Surg. 70:1238-45 (2000).
Another potential mechanism is the freezing and storage process itself. Although it has been previously shown that FC accelerates degeneration in a syngeneic rodent model, the direct impact of ice formation on elastic and collagenous fiber containing tissues could not be displayed conclusively using conventional visualization methods. 14. Legare, J. F., Lee, D. G., Ross, D. B., Circulation 102: III75-78 (2000). The development and application of multiphoton-induced autofluorescence and SHG imaging has allowed visualization of ECM alterations with submicron resolution. See Schenke-Layland, K., et al., Ann Thorac Surg 81:918-26 (2006); see also Schenke-Layland, K. et al., Ann Thorac Surg 83:1641-50 (2007)
Studies utilizing multiphoton imaging (MI) methodologies have demonstrated that conventional FC by controlled freezing is accompanied by serious alterations of extracellular matrix (ECM) structures. See Schenke-Layland, K., Madershahian, N., Riemann, I. et al., Ann Thorac Surg 81:918-26 (2006).
Vitreous cryopreservation (VC) has been identified as an alternative preservation approach that avoids ice formation and preserves ECM in vitro. See Song, Y. C., Khirabadi B. S., Lightfoot, F. G., Brockbank, K. G. M., Taylor, M. J., Nature Biotech 18: 296-299 (2000); see also Schenke-Layland, K., Xie, J., Hagvall, S. H. et al., Ann Thorac Surg 83:1641-50 (2007).
Vitrification means solidification, as in a glass, without ice crystal formation. Principles of vitrification are well known. Generally, the lowest temperature a solution can possibly supercool to without freezing is the homogeneous nucleation temperature Th, at which temperature ice crystals nucleate and grow, and a crystalline solid is formed from the solution. Vitrification solutions have a glass transition temperature Tg, at which temperature the solute vitrifies, or becomes a non-crystalline solid. Owing to the kinetics of nucleation and crystal growth, it is effectively impossible for water molecules to align for crystal formation at temperatures much below Tg. In addition, on cooling most dilute aqueous solutions to their glass transition temperature, Th is encountered before Tg, and ice nucleation occurs, which makes it impossible to vitrify the solution. In order to make such solutions useful in the preservation of biological materials by vitrification, it is therefore necessary to change the properties of the solution so that vitrification occurs instead of ice crystal nucleation and growth.
While it is generally known that high hydrostatic pressures raise Tg and lower Th, vitrification of most dilute solutions by the application of pressure is often impossible or impractical. In particular, for many solutions vitrifiable by the application of pressure, the required pressures cause unacceptably severe injury to unprotected biomaterials during vitrification thereof. While it is also known that many solutes, such as commonly employed cryoprotectants like DMSO, raise Tg and lower Th, solution concentrations of DMSO or similar solutes high enough to permit vitrification typically approach the eutectic concentration and are generally toxic to biological materials.